The quantitative determination of alkaline phosphatase and isoenzymes thereof in biological fluids, and particularly in human blood serum, has become very important in the diagnosis and treatment of various physical disorders. Such determination is very important in the detection of bone and liver diseases in view of elevated concentrations of alkaline phosphatase associated with such diseases. For example, elevated alkaline phosphatase concentrations are often associated with Paget's disease, osteosarcomas, osteomalacia, obstructive jaundice, hepatitis and the like. An early and rapid detection of such elevated alkaline phosphatase concentrations can then lead to rapid treatment of the causative conditions. As a result, various assay procedures have been developed over the years to provide a quantitative determination of alkaline phosphatase.
The disodium salt of p-nitrophenyl phosphate (PNPP) is known in the art as a substrate for alkaline phosphatase assays. However, the stability of this substrate is limited because the sodium ions of the substrate accelerate spontaneous nonenzymatic hydrolysis. In U.S. Pat. No. 3,425,912 (issued Feb. 4, 1969 to Deutsch et al), reagents for use in a solution assay of alkaline phosphatase include an amine salt of PNPP, a buffer (pH 4-11), such as disodium or sodium hydrogen carbonate, and a magnesium salt activator. These reagents are made and kept together in dry form until immediately before use. While dry, the reagents are reportedly stable and have a long shelf life.
It would be desirable to have a dry test element for measuring alkaline phosphatase and thereby avoid solution assays and the use of dry powders and the problems associated therewith. As is well known, dry elements have several advantages over solution assays, including the avoiding of mixing or reconstituting reagents, minimizing sample to sample contamination or inaccuracies, their suitability for use by less skilled personnel and fewer storage requirements.
However, it has been found that when the most common and preferred reagents used in the alkaline phosphatase assay (i.e. PNPP substrate and 2-amino-2-methyl-1-propanol (2A2M1P) buffer and phosphate acceptor) are incorporated into conventional dry test elements, such as those described in U.S. Pat. No. 3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al), a serious problem in pH control is encountered. Even when such elements are kept refrigerated for two weeks, a 30% loss in measured enzyme activity has been observed.
In measuring alkaline phosphatase in a liquid sample, it is essential that the pH of the sample be maintained at between about 9.7 and 10.5 for optimum enzyme activity. 2A2M1P (2-amino-2-methyl-1-propanol) is a common buffer and phosphate acceptor for alkaline phosphatase solution assays, but it has been found not to provide the needed buffering capacity in a dry element used for an alkaline phosphatase assay. When 2A2M1P is used as the buffer in a dry element, the pH tends to drift downward with time. This pH drift results in the serious loss in enzyme activity noted above.
It has been found that, in general, buffers have a pKa of 9 to 11.5 which are alkali metal or ammonium salts, can provide needed pH control in dry test elements. The use of a carbonate buffer in a dry test element for detecting alkaline phosphatase is described, for example, in U.S. Pat. No. 3,905,872 (issued Sept. 16, 1975 to Forgione). However, it has been found that the cation (e.g. sodium ions) from such buffers accelerate spontaneous nonenzymatic hydrolysis of the PNPP substrate. Such hydrolysis begins immediately when the reagents are coated to form an element and continues during storage. The end result of such substrate instability is an unwanted decline in the sensitivity of the dry element to alkaline phosphatase, which decline continues during storage.
Hence, there is a need in the art for a dry element for determining alkaline phosphatase in which the pH is easily controlled at optimum levels while simultaneously maintaining substrate stability and corresponding element sensitivity to the enzyme.